Motor proteins in Saccharomyces cerevisiae

1995 ◽  
Vol 73 (S1) ◽  
pp. 369-371 ◽  
Author(s):  
Susan S. Brown
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Key Words ◽  
Motor Proteins ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Temperature Sensitive ◽  
Related Protein ◽  
Yeast Saccharomyces Cerevisiae

A number of myosins have been identified in yeast (Saccharomyces cerevisiae), an organism ideally suited to dissecting out their different functions. We have learned that a temperature-sensitive defect in one of these myosins (Myo2p) can be partially overcome by overexpression of a kinesin-like protein (Smy1p). This raises the possibility of the involvement of microtubules in the same function as Myo2p. However, we have been unable to demonstrate that this is the case, either using nocodazole to depolymerize microtubules or by altering the nucleotide-binding site of Smy1p. Key words: myosin, kinesin-related protein, cytoskeleton.

Conformational changes at the nucleotide pocket of motor proteins monitored by electron paramagnetic resonance spectroscopy

2011 ◽  
Vol 83 (9) ◽  
pp. 1675-1684 ◽  
Author(s):  
Nariman Naber ◽  
Roger Cooke ◽  
Edward Pate
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Binding Site ◽  
Conformational Changes ◽  
Motor Proteins ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Nucleotide Site ◽  
Electron Paramagnetic

A fundamental goal in the field of motor proteins is to identify the conformational changes associated with the hydrolysis of the physiological substrate, ATP, and to define how these conformational changes are modulated by binding to the polymer track and translated into biologically useful movement. We have used electron paramagnetic resonance (EPR) spectroscopy to monitor conformational changes at the nucleotide-binding site of myosin- and kinesin-family motors. A novel set of nucleotide-analog EPR spin probes were synthesized and used to localize a spin moiety at the nucleotide site. This allows a reporter group to be placed with high specificity at the ATP binding site. Our results indicate that the nucleotide-binding site of myosin motors opens when the motor binds to its polymer roadway, actin, while that of kinesin closes on binding to microtubules (MTs). However, the transition is not all-or-none. There is instead an equilibrium between open and closed conformations. The different conformational changes in the two motor families can be correlated with differences in their biochemical cycles. Thus, we can now define the relationship between nucleotide-site structure, biochemistry and polymer binding for the two motors.

scholarly journals Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae.

1994 ◽  
Vol 125 (4) ◽  
pp. 825-842 ◽  
Author(s):  
S H Lillie ◽  
S S Brown
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Binding ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Polarized Growth ◽  
Related Protein ◽  
Active Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
Double Labeling ◽  
Unconventional Myosin

Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H. and S. S. Brown, 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smy1p and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization. The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain.

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

2021 ◽  
pp. 1-15
Author(s):  
Zengzhi Si ◽  
Yake Qiao ◽  
Kai Zhang ◽  
Zhixin Ji ◽  
Jinling Han
Keyword(s):  
Binding Site ◽  
Abiotic Stresses ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Nucleotide Binding ◽  
Regulatory Elements ◽  
Nucleotide Binding Site ◽  
Biotic And Abiotic Stresses ◽  
Encoding Genes

Sweetpotato, <i>Ipomoea batatas</i> (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that <i>IbNBS75</i>, <i>IbNBS219</i>, and <i>IbNBS256</i> respond to stem nematode infection; <i>Ib­NBS240</i>, <i>IbNBS90</i>, and <i>IbNBS80</i> respond to cold stress, while <i>IbNBS208</i>, <i>IbNBS71</i>, and <i>IbNBS159</i> respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

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